Trafficking and phosphorylation dynamics of AQP4 in histamine‐treated human gastric cells

Abstract

Background information . AQP4 (aquaporin 4) internalization and a concomitant decrease in the osmotic water permeability coefficient ( P f ) after histamine exposure has been reported in AQP4‐transfected gastric HGT1 cells. Results . In the present study we report that AQP4 internalization is followed by an increase in AQP4 phosphorylation. Histamine treatment for 30 min resulted in an approx. 10‐fold increase in AQP4 phosphorylation that was inhibited by 1 μM H89, a specific PKA (protein kinase A) inhibitor, but not by PKC (protein kinase C) and CK2 inhibitors. Moreover, measurement of PKA activity after 30 min of histamine treatment showed that PKA activity was approx. 3‐fold higher compared with basal conditions. AQP4 phosphorylation was prevented in cells treated with histamine for 30 min after pre‐incubation with PAO (phenylarsine oxide), an inhibitor of protein endocytosis. Using an endo—exocytosis assay we showed that, after histamine washed out, internalized AQP4 recycled back to the cell surface, even in cells in which de novo protein synthesis was inhibited by cycloheximide. Conclusions . Phosphorylation experiments, combined with immunolocalization studies, indicated that AQP4 phosphorylation is mediated by PKA and occurs subsequently to its internalization in late endosomes. We suggest that phosphorylation might be a mechanism involved in retaining AQP4 in a vesicle‐recycling compartment.